In connection with the diagnosis and treatment of certain health problems, it is most useful to identify and measure the various antibodies, drugs, proteins and other "macromolecules" in various body fluids. One of the most widely used techniques for making such determinations is the radioimmunoassay. The history of radioimmunoassay development is set forth, for example, in an article entitled "A Physicist In Biomedical Investigation", by Rosalind S. Yalow, Physics Today, October 1979, pages 25-29. The principal disadvantages of radioimmunoassays are their reliance on expensive and potentially hazardous reagents which possess a limited shelf life, the special handling and disposing procedures which are required for radioactive material, and the expensive instrumentation which is needed.
There are also several fluorescence-based immunoassay techniques which are currently in use or are undergoing clinical evaluation, with one of these techniques being described in a booklet entitled "Immuno-fluor", and subtitled "Quantitative Immunofluorescent Assay for Human Gamma Globulin", May 1978, and originating with Bio-Rad Laboratories. In one type of radiation and fluorescent immunoassay technique which has been proposed, relatively large carrier particles are coated with one of the active materials, usually the antibody, a sample to be tested is added to the solution, and the antigen in the sample is bound to the antibody. A fluorescently labeled antibody is then added to the mixture and binds to the antigen. The amount of fluorescently labeled antibody which is attached to the antigen, and is therefore bonded to the larger particles is directly proportional to the amount of the antigen in the sample. The excess fluorescently labeled antibody is then separated by standard centrifuging and decanting techniques. The remaining material consists substantially of the large particles and the attached (1) antibody, (2) antigen, and (3) fluorescently tagged antibody. The level of fluorescence of this residual material indicates the level of antigen present in the sample under test. This reaction is called a "sandwich" reaction because the antigen is sandwiched between the antibody coated to the carrier particle and the fluorescently labeled antibody.
In another immunoassay technique, carrier particles are also coated with an antibody. The coated particles, the sample with an unknown amount of antigen, and a known amount of tagged (either radioactive or fluorescent) antigen are placed in solution. The unknown antigen and the tagged antigen then complete for binding to the antibody. The carrier particles are then separated out of the solution and the amount of tagged antigen on the carrier particles is a measure of the amount of unknown antigen in the sample, the more tagged antigen bound to the carrier particles the less the unknown antigen in the sample. This type of reaction is called a competitive reaction, since the tagged antigen and unknown antigen compete for binding to the antibody which is attached to the carrier particles.
The foregoing procedures are employed in a number of radioactive-based and fluorescent-labeled assays. In each case, however, the analysis is made in the course of a reaction in which radioactive or fluorescent-tagged substances are either bound to larger particles or are shifted into solution from previous sites on the larger particles. Subsequently, the larger carrier particles are centrifuged, the residual liquid is decanted, and this is sometimes followed by additional purification steps in which liquid is added and another centifuging step is undertaken to insure the removal of any unbound component which might otherwise affect the assay signal obtained from the residue associated with the larger carrier particles.
While the prior assay techniques have proved very accurate, and are widely used, a large amount of time is spent in the separation steps; and in the case of the radioassay methods, the problem of handling the radioactive material is troublesome, and of course the undesired extra exposure to radiation is unfortunate, with its adverse health implications.
Accordingly, a principal object of the present invention is to provide an assay or analysis method which does not require the use of radioactive material, and also which does not require the physical separation of the carrier particles from the solution.